(1) Technical Field
The present invention relates to an automated apparatus for injecting a sample of gene, pigment, protein, peptide or drug into oocytes of Amphibia such as frog using a pipette-like needle. The present invention further pertains a method for injecting the sample of gene, pigment, protein, peptide or drug into the specified position of the amphibian oocytes, the amphibian oocytes with a guaranteed quality for injection of the sample, and a method for selling or assigning the amphibian oocytes into which the sample is injected at a specified position and depth.
(2) Background Art
Since oocytes of frogs such as Xenopus lavis, into which gene, pigment, protein, peptide or drug is injected, have a comparatively large size and can be obtained with a low cost and in large quantities, they are widely used for the purpose of confirming actions of pigment, protein, peptide and drug on viable cells, analysis of gene functions, and production of proteins as a gene product. To this purpose, the individual research scientists breed frogs by themselves and collect the oocytes.
Heretofore, in the injection of a sample such as gene, pigment, protein, peptide and drug into the oocytes of Amphibia such as frog, technicians manually injected with a pipette-like needle in which these samples are packed, into the oocytes under a microscopic observation using a manipulator. The pipette is mounted on the injector and a constant amount of sample is injected and exhaled into cells by an action of an oil pressure or an air pressure. Further, JP-A-5-192171 and JP-A-6-343478 disclose a method for injection and exhalation of a constant amount of sample into cells by applying a voltage. These prior arts disclose techniques for injection of sample by approaching needle manually to the cell under observing the cell microscopically.
The above manual injection of samples such as genes had a problem that a rate of oocytes, into which sample was successfully injected, was varied depending on skills and experiences of technicians. This was due to a difference in numbers of oocytes, into which the sample could be injected within a fixed time, depending on the individual technician, and as a result, a degree of denaturation of the sample was varied in a time-dependent manner among the technicians. It is an object of the present invention to provide a method to give a constant numbers of cells per hour to be treated independent of the skills and experiences of the technicians.
Further, the above described prior art did not take into the for unifying of the injection depth of samples into the oocytes at a constant depth. Consequently, it was difficult to control the depth even by a skilled technician. As a result, an injection of a sample into a specified cell organelle such as a nucleus had to depend on chance. It is another object of the present invention to provide an easier method for injecting a sample into a cellular organelle such as a nucleus, in which the injection can be controlled in a depth direction by unifying the injection depth of the sample.
Furthermore, in the above described prior art, since no consideration was given for memorizing information of cells in the injection of the sample, it was difficult to obtain a correlation between the information on the sample injection and the subsequent cell reaction. Consequently, it is further object of the present invention to provide a method for obtaining the correlation between them.
In addition, in the conventional techniques, the oocytes were prepared individually and thus a mass production or production on demand in good time was impossible.
Consequently, it is a further object of the present invention to provide the oocytes, into which a specified sample is injected, or the production, sale and transportation of the oocytes with a guaranteed injection of the specified sample at a constant position.
In order to solve the above problems, the present invention provides an apparatus for automatically injecting the sample such as gene, pigment, protein, peptide or drug into any constant position and any constant depth of the oocytes of Amphibia such as frogs by using a pipette-like needle.
Namely, the present invention provides an apparatus for microinjection of samples into amphibian oocytes comprising a tray for holding a plurality of the amphibian oocytes, an injection needle for injecting a sample into the said amphibian oocytes, a driving means for moving a relative position of the said tray to the said injection needle and a controlling means for controlling the said movement by inputting a depth of the said injection needle for the said tray or the said amphibian oocytes in the injection of the sample, and injecting the sample into the said amphibian oocytes at the said depth.
The present invention further provides a system for microinjection of samples into amphibian oocytes comprising a tray for holding a plurality of the amphibian oocytes, an injection needle for injecting sample into the said amphibian oocytes, a driving means for moving a relative position of the said tray to the said injection needle in a three dimensional direction, a controlling means for controlling the said movement, an information obtaining means for obtaining visual information on the said amphibian oocytes in the microinjection, and a memorizing means for accumulating the said information, and injecting the said sample into the said amphibian oocytes.
As a result, the sample can be injected rapidly into the plurality of amphibian oocytes at an almost constant depth.
Further, the tray has a plurality of wells having a cylindrical structure with a flat base or with a conical base having a maximum diameter of 105-150% of a diameter of the amphibian oocytes. As a result, the sample can be injected into the identical surface in about 80% of oocytes on the tray without applying any special means.
We have found that when mRNA was injected into the oocytes, in case that mRNA was injected into the animal hemisphere of the oocyte or mRNA was injected into the vegetal hemisphere, expression efficiency or the functional expression efficiency is different. Namely, in order to suppress a variation of the expression efficiency of the function of a protein in oocytes, it is important to collect the oocytes, in which mRNA is injected into the same hemispherical surface. In the present invention, it is possible to memorize information of cellular area in the injection of the sample and to induce easily a correlation between the information and the subsequent cell reaction.
Further, the present invention provides a method for automatic microinjection of samples into amphibian oocytes comprising,
using an apparatus having a tray for holding a plurality of the amphibian oocytes and an injection needle for injecting a sample into a plurality of the said amphibian oocytes,
a step for setting a depth of the said injection needle for the said tray or the said amphibian oocytes at the first depth,
a step for injecting the sample into the first oocyte in a plurality of the said amphibian oocytes using the said injection needle at the said first depth,
a step for automatically moving a relative position of the said tray to the said injection needle, and
a step for subsequently injecting the sample into the second oocyte in a plurality of the said amphibian oocytes by using the said injection needle to the said first depth.
Furthermore, the present invention provides a method for automatic microinjection of samples into amphibian oocytes comprising,
using an apparatus having a tray for holding a plurality of the amphibian oocytes and an injection needle for injecting a sample into a plurality of the said amphibian oocytes,
a step for injecting the sample into the first oocyte in a plurality of the said amphibian oocytes using the said injection needle,
a step for moving a relative position of the said tray to the said injection needle,
a step for subsequently injecting the sample into the second oocyte in a plurality of the said amphibian oocytes using the said injection needle,
a step for obtaining a condition of oocyte in the injection of the said sample as a visual information, and
a step for accumulating the said visual information.
According to the invention of the above described apparatus or method for injection of the sample into the amphibian oocytes, the present invention further provides a plurality of the amphibian oocytes wherein the sample is injected under a substantially equal condition in the injection depth of the sample.
The present invention also provides the said amphibian oocytes wherein the sample is further injected under a substantially same condition in the injection position of the sample.
In addition, the present invention provides the following methods.
A method for preparation of a group of the amphibian oocytes injected with a sample comprising,
using an apparatus having a tray for holding a plurality of the amphibian oocytes and an injection needle for injecting sample into a plurality of the said amphibian oocytes,
moving a relative position of the said injection needle to the said tray,
injecting the sample into each of a plurality of the said respective amphibian oocytes using the said injection needle,
obtaining each visual information of the said amphibian oocytes in the injection, and
collecting a plurality of the oocytes in which the sample is injected into the animal hemisphere of the oocyte, or a plurality of the oocytes in which the sample is injected into the vegetal hemisphere of the oocyte in a plurality of the said amphibian oocytes based on the said visual information.
A method for selling or assigning a plurality of the amphibian oocytes comprising selling or assigning as a set a plurality of the amphibian oocytes, into which the sample is injected under a substantially equal condition in the injection depth of the sample, and attaching the information on the injection of the sample into a plurality of the amphibian oocytes to the set.
In addition, a method for selling or assigning a plurality of the amphibian oocytes comprising putting a plurality of the amphibian oocytes into which the sample is injected under a substantially equal condition in the injection depth of the sample into a vessel, and attaching a label on which the information on the injection of the sample into a plurality of the said amphibian oocytes is described to the said vessel.
In this connection, the information on the injection of the sample into a plurality of the amphibian oocytes relates to at least any one of a date and time of the injection, a term for guarantee of a quality, a position where the sample is injected, a depth at which the sample is injected and a probability of expression.
As a result, according to the present invention, the oocytes having substantially identical condition in relation to the injections as well as their information can be obtained.